Cytomegalovirus viral load within blood increases markedly in healthy people over the age of 70 years.

BACKGROUND: Cytomegalovirus (CMV) is a highly prevalent herpesvirus, which maintains lifelong latency and places a significant burden on host immunity. Infection is associated with increased rates of vascular disease and overall mortality in the elderly and there is an urgent need for improved understanding of the viral-host balance during ageing. CMV is extremely difficult to detect in healthy donors, however, using droplet digital PCR of DNA from peripheral blood monocytes, we obtained an absolute quantification of viral load in 44 healthy donors across a range of ages. RESULTS: Viral DNA was detected in 24 % (9/37) of donors below the age of 70 but was found in all individuals above this age. Furthermore, the mean CMV load was only 8.6 copies per 10,000 monocytes until approximately 70 years of age when it increased by almost 30 fold to 249 copies in older individuals (p < 0.0001). CMV was found within classical CD14+ monocytes and was not detectable within the CD14-CD16+ subset. The titre of CMV-specific IgG increased inexorably with age indicating that loss of humoral immunity is not a determinant of the increased viral load. In contrast, although cellular immunity to the structural late protein pp65 increased with age, the T cell response to the immediate early protein IE1 decreased in older donors. CONCLUSION: These data reveal that effective control of CMV is impaired during healthy ageing, most probably due to loss of cellular control of early viral reactivation. This information will be of value in guiding efforts to reduce CMV-associated health complications in the elderly.

Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase.

Macrophages exposed to macrophage colony-stimulating factor acquire the capacity to suppress T cell proliferation; this effect is associated with de novo expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). We have purified IDO and tested its activity in in vitro models of T cell activation. IDO was able to inhibit proliferation of CD4(+) T lymphocytes, CD8(+) T lymphocytes, and natural killer (NK) cells; proliferation of B lymphocytes was not affected. The inhibitory role of tryptophan and of its catabolites was then tested. In the presence of tryptophan, only L-kynurenine and picolinic acid inhibit cell proliferation. In a tryptophan-free medium cell proliferation was not affected. In the absence of tryptophan inhibition induced by L-kynurenine and picolinic acid was observed at concentrations below the lowest concentration that was effective in the presence of tryptophan, and quinolinic acid acquired some inhibitory capacity. Inhibition of cell proliferation induced by the tryptophan catabolites resulting from IDO activity was selective, applying only to cells undergoing activation. Resting cells were not affected and could subsequently activate normally. We suggest that IDO exerts its effect on cell proliferation by (i) starting the cascade of biochemical reactions that produce the three catabolites and by (ii) enhancing their inhibitory potential by depriving the extracellular microenvironment of tryptophan.

Homeostatic Cytokines Drive Epigenetic Reprogramming of Activated T Cells into a "Naive-Memory" Phenotype.

Primary stimulation of T cells is believed to trigger unidirectional differentiation from naive to effector and memory subsets. Here we demonstrate that IL-7 can drive the phenotypic reversion of recently differentiated human central and effector memory CD8+ T cells into a naive-like phenotype. These "naive-revertant" cells display a phenotype similar to that of previously reported stem cell memory populations and undergo rapid differentiation and functional response following secondary challenge. The chromatin landscape of reverted cells undergoes substantial epigenetic reorganization with increased accessibility for cytokine-induced mediators such as STAT and closure of BATF-dependent sites that drive terminal differentiation. Phenotypic reversion may at least partly explain the generation of "stem cell memory" CD8+ T cells and reveals cells within the phenotypically naive CD8+ T cell pool that are epigenetically primed for secondary stimulation. This information provides insight into mechanisms that support maintenance of T cell memory and may guide therapeutic manipulation of T cell differentiation.

CD117 (c-Kit) Is Expressed During CD8+ T Cell Priming and Stratifies Sensitivity to Apoptosis According to Strength of TCR Engagement.

CD117 (cKit) is the receptor for stem cell factor (SCF) and plays an important role in early haemopoiesis. We show that CD117 is also expressed following priming of mature human CD8+ T cells in vitro and is detectable following primary infection in vivo. CD117 expression is mediated through an intrinsic pathway and is suppressed by IL-12. Importantly, the extent of CD117 expression is inversely related to the strength of the activating stimulus and subsequent engagement with cell-bound SCF markedly increases susceptibility to apoptosis. CD117 is therefore likely to shape the pattern of CD8+ T cell immunodominance during a primary immune response by rendering cells with low avidity for antigen more prone to apoptosis. Furthermore, CD117+ T cells are highly sensitive to apoptosis mediated by galectin-1, a molecule commonly expressed within the tumor microenvironment, and CD117 expression may therefore represent a novel and potentially targetable mechanism of tumor immune evasion.

Arginase 2 is expressed by human lung cancer, but it neither induces immune suppression, nor affects disease progression.

In human prostate cancer, Arginase 2 (ARG2) and nitric oxide synthase (NOS) are concomitantly expressed by tumor cells, and induce tumor immune escape via peroxynitrite-dependent Tyrosine nitrosylation. Since there were no data regarding this immune suppressive mechanism in other tumor types, and an evaluation of its clinical relevance in human tumors had still to be provided, we have investigated presence and clinical relevance of ARG2 and NOS expression in lung cancer. No evidence of NOS expression was found, no significant NOS enzymatic activity was detected. Instead, ARG2 protein was expressed by tumor cells. In a cohort of 120 patients, the amount of ARG2-positive tumor cells was significantly higher in small cell lung cancers (SCLC) than in non-small cell lung cancers (NSCLC). Large cell undifferentiated carcinomas had twice ARG2 than the other NSCLC subtypes. ARG2 expression was increased in Grade 3 tumors, as compared to Grades 1 and 2. However, no relationship was found with tumor size and stage, and with patient survival. Indeed, the enzyme was active, since the Arginine catabolite Ornithine was produced, but Arginine depletion was not attained. In addition, nitrotyrosine was not found in tumor tissue. Accordingly, when tumor cells isolated from lung cancer were incubated with activated autologous T cells, no inhibition of proliferation was detected. Our results indicate that ARG2 is expressed in lung cancer, but it does not induce tumor immune escape and does not affect disease progression, most probably due to the lack of concomitant NOS expression.

Biomechanical properties of human T cells in the process of activation based on diametric compression by micromanipulation.

A crucial step in enabling adoptive T cell therapy is the isolation of antigen (Ag)-specific CD8+ T lymphocytes. Mechanical changes that accompany CD8+ T lymphocyte activation and migration from circulating blood across endothelial cells into target tissue, may be used as parameters for microfluidic sorting of activated CD8+ T cells. CD8+ T cells were activated in vitro using anti-CD3 for a total of 4 days, and samples of cells were mechanically tested on day 0 prior to activation and on day 2 and 4 post-activation using a micromanipulation technique. The diameter of activated CD8+ T cells was significantly larger than resting cells suggesting that activation was accompanied by an increase in cell volume. While the Young's modulus value as determined by the force versus displacement data up to a nominal deformation of 10% decreased after activation, this may be due to the activation causing a weakening of the cell membrane and cytoskeleton. However, nominal rupture tension determined by compressing single cells to large deformations until rupture, decreased from day 0 to day 2, and then recovered on day 4 post-activation. This may be related to the mechanical properties of the cell nucleus. These novel data show unique biomechanical changes of activated CD8+ T cells which may be further exploited for the development of new microfluidic cell separation systems.

Eosinophil granulocytes account for indoleamine 2,3-dioxygenase-mediated immune escape in human non-small cell lung cancer.

Indoleamine 2,3-dioxygenase (IDO), a catabolizing enzyme of tryptophan, is supposed to play a role in tumor immune escape. Its expression in solid tumors has not yet been well elucidated: IDO can be expressed by the tumor cells themselves, or by ill-defined infiltrating cells, possibly depending on tumor type. We have investigated IDO expression in 25 cases of non-small cell lung cancer (NSCLC). Using histochemistry and immunohistochemistry, we found that IDO was expressed not by tumor cells, but by normal cells infiltrating the peritumoral stroma. These cells were neither macrophages nor dendritic cells, and were identified as eosinophil granulocytes. The amount of IDO-positive eosinophils varied in different cases, ranging from a few cells to more than 50 per field at x200 magnification. IDO protein in NSCLC was enzymatically active. Therefore, at least in NSCLC cases displaying a large amount of these cells in the inflammatory infiltrate, IDO-positive eosinophils could exert an effective immunosuppressive action. On analyzing the 17 patients with adequate follow-up, a significant relationship was found between the amount of IDO-positive infiltrate and overall survival. This finding suggests that the degree of IDO-positive infiltrate could be a prognostic marker in NSCLC.

Differential regulation of spontaneous and immune complex-induced neutrophil apoptosis by proinflammatory cytokines. Role of oxidants, Bax and caspase-3.

Neutrophil apoptosis represents a crucial step in the mechanisms governing the resolution of neutrophilic inflammation. Several soluble mediators of inflammation modulate neutrophil survival, retarding their apoptosis, whereas neutrophil activation by immune complexes (IC) results in the acceleration of apoptosis. To investigate neutrophil fate at the site of inflammation, we studied the effects of interleukin (IL)-2, IL-6, IL-8, IL-15, GM-CSF, and fMLP on spontaneous and IC-induced neutrophil apoptosis and the mechanisms regulating the survival of these cells. Spontaneous apoptosis was inhibited by GM-CSF, IL-6, and IL-15, but only GM-CSF overturned IC-induced apoptosis. No role of oxidants on the modulation of IC-dependent apoptosis was found. Indeed, fMLP or GM-CSF augmented the IC-dependent oxidative response, whereas the other compounds were ineffective. CGD neutrophils showed low levels of spontaneous apoptosis, but when exposed to IC, underwent a sharp increment of the apoptotic rate in a GM-CSF-inhibitable manner. Conversely, the expression of the proapoptotic protein Bax in 18-h aged neutrophils was down-regulated by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a nearly threefold Bax up-regulation, which was completely reversed only by GM-CSF. Accordingly, the spontaneous activity of caspase-3 was inhibited by GM-CSF, IL-6, and IL-15. Furthermore, IC induced a sharp increment of enzymatic activity, and only GM-CSF inhibited the IC-dependent acceleration. Our results show that apoptosis of resting and IC-activated neutrophils is regulated differently, GM-CSF being the most potent neutrophil antiapoptotic factor. The results also unveil the existence of an oxidant-independent, Bax- and caspase-3-dependent, intracellular pathway regulating neutrophil apoptosis.

Delayed neutrophil apoptosis induced by synovial fluid in rheumatoid arthritis: role of cytokines, estrogens, and adenosine.

The fate of neutrophils at sites of inflammation, where these cells are likely exposed to both anti- and proapoptotic influences, needs to be clarified. To investigate this issue, we studied the survival of neutrophils in the presence of articular fluids from RA joints before and after immune complex activation. Eight of eleven samples of RA synovial fluid studied were found to inhibit spontaneous and immune complex-stimulated neutrophil apoptosis. No relationships were found between GM-CSF and TNF-alpha concentrations measured on each sample of synovial fluid studied and the levels of neutrophil apoptosis detectable in the presence of the same synovial fluid. Furthermore, no activity on neutrophil survival was observed at either physiologic or pharmacologic concentrations of estradiol. On the contrary, the synovial fluid anti-apoptotic activity correlates (r(2) = 0.8818, p < 0.0001) with the adenosine detected at concentrations in each sample ranging from 18.7 to 52.4 microM. Finally, synovial fluids were incapable of interfering with neutrophil activation evaluated as superoxide anion production. Our results suggest that the microenvironment of rheumatoid synovial fluid is a proinflammatory milieu responsible for the in loco persistence of activated and long-surviving neutrophils.

Taurine prevents apoptosis induced by high ambient glucose in human tubule renal cells.

BACKGROUND: Hyperglycemia selectively triggers apoptosis in tubule and endothelial cells. Taurine, a conditionally essential amino acid, is abundant in several tubule segments, but its role has not been defined fully. It can serve as an osmolyte or as an endogenous antioxidant. Taurine metabolism is altered in diabetes mellitus, with extracellular and intracellular pools reduced. It is still unknown whether taurine can play a role as a protective agent in apoptosis induced by high glucose in tubular cells. METHODS: Apoptosis (by annexin V binding and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling method), cellular reactive oxygen species (ROS) formation (by fluorescent probe 2'-7' dichlorofluorescin diacetate and FACScan flow cytometry), and Bcl-2 and Bax proteins (by immunostaining) were studied in a human proximal tubular cell line (HK-2) grown in a medium with physiologic (5.5 mM) or high (30 mM) glucose concentrations for 48 hours. In separate experiments, taurine (3-24 mM) was added to the media. RESULTS: The exposure of human tubule cells to 30 mM glucose for 48 hours resulted in a significant increase in apoptosis compared with 5.5 mM glucose (35 +/- 8% vs. 6 +/- 3%, p < 0.001). Thirty mM mannitol failed to induce the effects of high glucose. High glucose-mediated apoptosis was associated with a decrease in the expression of Bcl-2 (-87%) and a twofold increase in the expression of Bax protein. Taurine had a dose-dependent effect in preventing high-glucose-induced apoptosis (-78%, p < 0.001 at 24 mM). Moreover, with taurine, intracellular ROS decreased by 34% (p < 0.05), and changes in intracellular ROS formation induced by taurine at 24 hours predicted the variations in the apoptotic index at 48 hours (r = 0.87, p < 0.02). Other antioxidants, such as glutathione and N-acetylcysteine, also attenuated the high glucose-induced apoptosis. CONCLUSION: These results demonstrate that taurine attenuates hyperglycemia-induced apoptosis in human tubular cells via an inhibition of oxidative stress. Taurine might act as an endogenous antioxidant in tubule cells and could exert a beneficial effect in preventing tubulointerstitial injury in diabetic nephropathy.

Natural killer cells infiltrating human nonsmall-cell lung cancer are enriched in CD56 bright CD16(-) cells and display an impaired capability to kill tumor cells.

BACKGROUND: Despite natural killer (NK) cells being originally identified and named because of their ability to kill tumor cells in vitro, only limited information is available on NK cells infiltrating malignant tumors, especially in humans. METHODS: NK cells infiltrating human nonsmall cell lung cancers (NSCLC) were analyzed with the aim of identifying their potential protective role in an antitumor immune response. Both relevant molecule expression and functions of NK cells infiltrating NSCLC were analyzed in comparison with autologous NK cells isolated from either peritumoral normal lung tissues or peripheral blood. RESULTS: The CD56 bright CD16(-) NK cell subset was consistently observed as being highly enriched in tumor infiltrate and displayed activation markers, including NKp44, CD69, and HLA-DR. Remarkably, the cytolytic potential of NK cells isolated from cancer tissues was lower than that of NK cells from peripheral blood or normal lung tissue, whereas no difference was observed regarding their capability of producing cytokines. With regard to their localization within tumor, NK cells were found in tumor stroma, whereas they were not in direct contact with cancer cells. CONCLUSIONS: For the first time NK cells infiltrating NSCLC have been characterized and it is shown that they are mainly capable of producing relevant cytokines rather than exerting direct cancer cell killing.

Targeting tumor-related immunosuppression for cancer immunotherapy.

Tumors produce several factors, such as Prostaglandins (PGs), Interleukin (IL)-10, Vascular Endothelial Growth Factor (VEGF) and Transforming Growth Factor (TGF)-beta, which may directly or indirectly inhibit the immune response and may hamper immunotherapy. Furthermore, cells of innate or adaptive immunity, recruited by tumor-derived factors, may contribute in immunosuppression. Regulatory T (Treg) cells such as the "naturally occurring" CD4(+)/CD25(+) Treg and the IL-10-induced Tr1 cells are major players in this arena. Paradoxically Treg cells are stimulated by IL-2, which is used in tumor immunotherapy. Treg cells suppress T cell responses through soluble factors or by contact-dependent mechanisms, such as the Cytotoxic T Lymphocyte Antigen (CTLA)-4-mediated induction of Indoleamine 2,3-Dioxygenase (IDO) in dendritic cells (DC). IDO inhibits T cell responses by depleting Tryptophan and producing Kynurenine, which is toxic to lymphocytes. Macrophages, granulocytes or myeloid suppressor cells (MSC) suppress immunity by other enzymatic mechanisms, involving Arginase and Nitric Oxide Synthase (NOS). Subversion of tumor immunosuppression is required for successful immunotherapy. Attempts to block or eliminate Treg cells have been made by the use of chemotherapy, anti-CD25 or anti-CTLA-4 antibodies, IL-2-toxin chimeric proteins or Glucocorticoid-induced TNF-like Receptor (GITR) and CD134/OX-40 ligands. Tumor cells genetically modified to secrete IL-21 (an immune-stimulatory "IL-2-like" cytokine, which is not involved in immune regulation) cured experimental metastases in combination with anti-CD25 monoclonal antibodies (mAbs). Also strategies aimed at blocking enzyme-based immune-suppressive mechanisms are suitable, as suggested by experimental evidences in mouse tumor models.

The tryptophan catabolite L-kynurenine inhibits the surface expression of NKp46- and NKG2D-activating receptors and regulates NK-cell function.

Tryptophan (Trp) catabolism mediated by indoleamine 2,3-dioxygenase (IDO) plays a central role in the regulation of T-cell-mediated immune responses. In this study, we also demonstrate that natural killer (NK)-cell function can be influenced by IDO. Indeed, l-kynurenine, a Trp-derived catabolite resulting from IDO activity, was found to prevent the cytokine-mediated up-regulation of the expression and function of specific triggering receptors responsible for the induction of NK-cell-mediated killing. The effect of l-kynurenine appears to be restricted to NKp46 and NKG2D, while it does not affect other surface receptors such as NKp30 or CD16. As a consequence, l-kynurenine-treated NK cells display impaired ability to kill target cells recognized via NKp46 and NKG2D. Instead, they maintain the ability to kill targets, such as dendritic cells (DCs), that are mainly recognized via the NKp30 receptor. The effect of l-kynurenine, which is effective at both the transcriptional and the protein level, can be reverted, since NK cells were found to recover their functional competence after washing.

Block of Stat-1 activation in macrophages phagocytosing bacteria causes reduced transcription of CIITA and consequent impaired antigen presentation.

The IFN-gamma-induced HLA class II expression in human macrophages was drastically reduced after phagocytosis of Escherichia coli. HLA class II down-modulation depended on phagocytosis of bacteria and could not be reproduced by phagocytosis of inert particles or by treatment with lipopolysaccharide. Study of the kinetics and molecular analysis showed that class II molecules and corresponding mRNA were up-regulated at 6 h after phagocytosis of bacteria. Subsequently, a progressive reduction of mRNA occurred, and, at 72 h, as little as 25% of the class II mRNA level of IFN-gamma-treated control cells was found. This was due to reduced transcription of the class II transcriptional activator CIITA, as a consequence of reduced immediate-early inducible factor (IRF-1) and particularly of reduced phosphorylated Stat-1 homodimers, nuclear factors both necessary for optimal triggering of the CIITA promoter. Failure to sustain IFN-gamma-induced CIITA up-modulation during phagocytosis of bacteria had functional implications, as human macrophages could not adequately process and present antigenic peptides to HLA-DR-restricted antigen-specific T cells. This is the first evidence that phagocytosis of bacteria can down-modulate HLA class II expression in normal human macrophages by acting at the level of expression of CIITA.

Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors.

Killer immunoglobulin-like receptor (KIR) ligand incompatibility in the graft-versus-host direction was demonstrated to be associated with improved outcome in patients given haploidentical, T-cell-depleted hematopoietic stem cell transplants (HSCTs). The goal of this study was to evaluate whether that observation could be generalized for patients receiving unmanipulated HSCTs from unrelated donors (URD). One hundred thirty patients with hematologic malignancies entered the study. Graft-versus-host disease (GVHD) prophylaxis was uniform and consisted of cyclosporin, short-term methotrexate, and pretransplantation antithymocyte globulin (ATG). Patients were divided into those with (n = 20) and those without (n = 110) KIR ligand incompatibility with respect to their donors. At 4.5 years patients with KIR ligand incompatibility had higher probability of overall survival (87% versus 48%, P =.006) and disease-free survival (87% versus 39%, P =.0007) compared with those without KIR ligand incompatibility. Transplant-related mortality for the 2 groups equaled 6% and 40% (P =.01), respectively. Relapse rates for patients receiving transplants from a donor with or without KIR ligand incompatibility were 6% and 21%, respectively (P =.07). All patients with myeloid malignancies receiving transplants from KIR ligand-disparate donors (n = 13) are alive and disease free. These data indicate that natural killer (NK) cell alloreactivity is associated with better outcome after URD-HSC transplantation when ATG is used as part of GVHD prophylaxis.

A T-cell epitope encoded by a subset of HLA-DPB1 alleles determines nonpermissive mismatches for hematologic stem cell transplantation.

The importance of HLA-DPB1 matching for the outcome of allogeneic hematologic stem cell (HSC) transplantation is controversial. We have previously identified HLA-DPB1*0901 as a target of cytotoxic T cells mediating in vivo rejection of an HSC allograft. Here we show that HLA-DPB1*0901 encodes a T-cell epitope shared by a subset of DPB1 alleles that determines nonpermissive mismatches for HSC transplantation. Several T-cell clones obtained from the patient at the time of rejection showed HLA-DP restricted recognition of allogeneic targets expressing HLA-DPB1*0901, *1001, *1701, *0301, *1401, and *4501, but not other alleles. Based on these findings, we developed an algorithm for prediction of nonpermissive HLA-DPB1 mismatches. Retrospective evaluation of 118 transplantations showed that the presence of nonpermissive HLA-DPB1 mismatches was correlated with significantly increased hazards of acute grade II to IV graft-versus-host disease (HR = 1.87, P =.046) and transplantation-related mortality (HR = 2.69, P =.027) but not relapse (HR = 0.98, P =.939), as compared with the permissive group. There was also a marked but statistically not significant increase in the hazards of overall mortality (HR = 1.64, P =.1). These data suggest that biologic characterization of in vivo alloreactivity can be a tool for definition of clinically relevant nonpermissive HLA mismatches for unrelated HSC transplantation.